Camera and Ikaros karyotyping software implemented on a Dell Optiplex GX1 (MetaSystems, Heidelberg, Germany). Locus-specific FISH analysis of interphase nuclei and metaphase spreads was carried out with the following directly fluorescent labeled probe sets. As I work on the karyotyping of a species of earthworm, I would like to know if. Worked with human chromosomes using Metasystems IKAROS software [am.
Physiol Mol Biol Plants. 2011 Jul; 17(3): 313–316.
Published online 2011 Jun 29. doi: 10.1007/s12298-011-0072-8
PMID: 23573024
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Abstract
Podophyllum hexandrum is an important high altitude medicinal plant from Himalaya. Somatic chromosomes of this species were studied to delineate and physical mapping of repetitive rDNA sites to provide landmarks in chromosome identification. The karyotype formula of this species was found to be 6m + 2sm + 2st + 2t with secondary constriction in the chromosome 1 and 7. The FISH analysis of rDNA sites showed 4 sites for 18S rDNA and 2 sites for 5S rDNA. The chromosome number 1, 2, 5 and 6 can be identified based on 18S rDNA sites in their short arm and chromosome 1 and 2 can be identified by 5S rDNA site in the centromere region. The estimated genome size of this plant is 16.07 pg (1C).
Keywords: Podophyllum hexandrum, FISH, Flow cytometry, Genome size, rDNA localization
Introduction
Podophyllum hexandrum (Himalayan Mayapple) of family Berberidaceae is an important high altitude medicinal plant of Western Himalaya (found at 2,800–4,000 M). The rhizomes of P. hexandrum yield several lignans which possess antitumor activity. Podophyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. (Giri and Narasu ). The utilization of in situ hybridization technology is of particular interest those engaged in chromosome walking and genome mapping projects. FISH has been used in many plants to identify chromosome accurately using species specific repetitive sequences, ribosomal genes and unique genes. Because of the universal occurrence and redundancy, the ribosomal gens are great value for karyotype analysis and comparative genome analysis. FISH techniques using fluorescence allows the visualization of multigene families such as 5S and 18S-5.8S-26S ribosomal RNA genes for their localization on the chromosomes. In some lower plants, such as Marchantia polymorpha, 5S rDNAs are encoded within the 45S rDNA repeat units (Sone et al. ) in contrast to the arrangement in the chromosomes of higher plants, where 45S and 5S rDNA are physically separate and map independently. These two classes of rRNA genes co-localize on the chromosomes bearing the nucleolar organizing regions. The high copy number and tandem organization of these genes together provide useful markers for chromosome identification and karyotyping. The copy number of rDNA repeats can vary from hundreds to thousands of copies, per genome (Weider et al. 2005). Hence rDNA clusters can be easily visualized by FISH, which makes them good targets for initial studies on physical genome mapping. Physical localization and multiple copy of the above genes have been located in the chromosomes of Pinus (Liu et al. ; Cai et al. ), Triticum (Mukai et al. 1990) Brassica (Armstrong et al. 1998), Plantago (Dhar et al. ), Artemisia (Hoshi et al. 2006), Pseudotsuga (Amarsinghe and Carlson ), Lupinus (Naganowska and Zielinska ), Trifolium (Ansari et al. 1999). Since chromosome identification constitutes the first step in genetic manipulation of a species, the present investigation was therefore undertaken to provide meaningful landmarks to facilitate unequivocal chromosome identification of individual chromosomes employing DNA : DNA in situ hybridization.
Materials and methods
Preparation of chromosome spread
Growing root tips of P. hexandrum from the seeds were excised and pretreated with 0.1 % colchicine at 4 °C for 4 h for metaphase arrest and subsequently fixed in Carnoy’s fixative for 24 h and stored in 70 % ethanol at 4 °C afterwards. The fixative from the root tips was removed by washing in 1X enzyme buffer (citric acid, trisodium citrate buffer); and then macerated in enzyme solution at 37 °C (Mixture of 2 % w/v cellulase and 3 % v/v pectinase from Aspergilus niger) until the root tips get softened. The softened root tips were again washed with 1X enzyme buffer, followed by transfer to 45 % acetic acid. The chromosome spreads were prepared by conventional squashing.
Preparation of probe
Genomic DNA of P. hexandrum was isolated from the leaves, using CTAB method and used as a template for PCR amplification of rDNA. 18S rDNA was amplified using primers 5′-AACGGCTACCACATCCAAGG-3′ and 5′-GGTGGTGCCCTTCCGTCAAT-3′, whereas 5S rDNA was amplified using primers using 5′-CGATCATACCAGCACTAACG-3′ and 5′-TACTACTCTCGCCCAAGCAC-3′ (Kitamura et al. 2000). The PCR products were separated in 1.5 % agarose gels and the rDNA band, recovered and purified using GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The purified rDNA was labeled using FISH Tag DNA kit (Molecular Probes, Invitrogen), with Alexa Fluor 594 dye (Molecular Probes, Invitrogen) according to the manufacturer’s instructions.
![Ikaros karyotyping software reviews Ikaros karyotyping software reviews](/uploads/1/2/6/2/126248434/240020084.jpg)
Pretreatment and hybridization
Pretreatment of fixed chromosome was carried out for probe hybridization using method developed by Hesslop & Harrison with minor modification. The slides were then dehydrated in a graded ethanol series, and air dried. Double sided adhesive in situ frames (Eppendorf, Germany); were applied to the marked area on each slide. The hybridization mixture was prepared containing 10 μl (1 μg) of labeled DNA probe, 65 μl of formamide, 10 μl of 20X SSC and 15 μl of nuclease free water. This hybridization mixture was then denatured at 72 °C, for 5 min, and then kept in ice for 3 min. The denatured hybridization mixture was then applied to the marked area on the slide, and covered with a plastic coverslip adhering to the in situ frame. The chromosomes along with the hybridization mixture were denatured, by denaturing the slide at 72 °C on an in situ adapter (Eppendorf, Germany); for 5 min, followed by 8 h incubation in a humid chamber at 37 °C. The slides were then washed with 2X SSC, for 5 min twice, at 40 °C, followed by a dip in autoclaved distilled water. The slides were then kept for air drying in dark. It was counterstained with DAPI with Prolong Gold Antifade reagent (Invitrogen), and observed under Ziess Fluorescent Microscope (Ziess AxioImager. M1), equipped with CCD camera. Images were captured, using appropriate filter. The merging of fluorescent signals from the probe and the counter stain was done using ISIS software (Metasystems).
Genome size estimation
Genome size estimation was done by Flow Cytometry. Approximately 50 mg fresh green tissue from the leaf of P. hexandrum seedlings was excised and placed in a sterile plastic petriplate, on ice, along with 50 mg fresh green tissue from the leaf of internal standard i.e. Vicia faba L. ‘Inovec’ (Doležel et al. ). The leaf tissue was chopped into small fragments in 1 ml of ice cold nuclei isolation buffer (MgSO4 buffer). The nuclei were obtained through filtration and centrifugation. Fluorochrome PI was added along with RNase-A at 50 mg ml− concentration. The stained nuclear suspension was analyzed on FACS station, (Becton Dickinson FACSCalibur) and the data was analyzed using Becton Dickinson CellQuest Pro software. For karyotype analysis, chromosome images were captured by a CCD camera, on Ziess Axioimager, using Ikaros software (Metasystems). The average of chromosome length, chromosome arm ratio and position of hybridization signal was recorded, using Isis and Ikaros software. The chromosome arm ratio and arm length (r = p/q), were used to classify the chromosomes.
Results and discussion
Metaphase Chromosomes in P. hexandrum (2n = 12) are large in size, indicating towards a larger genome size, which was found to be 32.25 pg (2C content), using Vicia faba L. ‘Inovec’ (2C = 26.90), (Doležel et al. ) as internal standard. Three metaphase plates were used for chromosome measurement and classification. The classification system proposed by Levan et al. (1964) was used to classify the karyotype, in which the arm ratio (r = q/p), is used to classify karyotype (Moscone et al. ), in which the chromosomes are classified as m, metacentric (r = 1.00–1.69); sm submetacentric (r = 1.70–2.99); st, subtelocentric (r = 3.00–7.00); and t, telocentric (r > 7). The karyotype formula of P. hexandrum was found to be 6m + 2 sm + 2 st + 2 t. Four 18S signals identified (Fig. 1.), two of which are present on the metacentric chromosomes no. 1 & 2, towards the subtelomeric region; and are very light, whereas two very strong signals are found just near the centromeres, on the short arms of submetacentric chromosomes no. 5 & 7; whereas two very bright signals are found on the centromeres metacentric chromosomes no. 1 & 2. The respective positions are apparent in the ideogram (Fig. 2).
FISH based localization of two rDNA sites in P. hexandruma Metaphase chromosomes with 5S rDNA signals b Interphase with 5S rDNA signals c Metaphase chromosomes with 18S rDNA signals d Interphase with 18S rDNA signals (Bar = 10 μm)
Karyo-Ideogram showing the karyotype of P. hexandrum somatic chromosome and the position of hybridization signals from 5S and 18 S regions
Acknowledgement
We thank financial assistance from Department of Biotechnology, Govt. of India.
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